Apparatus for carrying out biochemical reactions

ABSTRACT

An apparatus for carrying out biochemical reactions in microtiter plates includes a device (42, 43) for receiving and holding microtiter plate (44) to be processed, the microtiter plate being provided with a plurality of wells and each well having a bottom outlet, and a movably mounted device (47) for the application of gas pressure to the wells of the microtiter plate (44), the pressure device including a sealing collar (48) and being movable between a position where it leaves the upper part of the microtiter plate accessible, and a position where it sealingly contacts the microtiter plate through the sealing collar (48) so that a gas pressure may be applied to the inlets of the microtiter plate wells for pressing out liquid present therein through the bottom outlet of each well.

The present invention is related to automation of biochemical reactions,and more particularly to the treatment of a plurality of samples inmicrotiter plates or similar processing plates in molecular biologicalmethods.

Molecular biology is a rapidly growing field of science but since it isa fairly young discipline a great number of basic methods are still donemanually, or at best semi-automatically. This being the case in spite ofthe fact that many of the procedures utilized are based on handling agreat number of samples, in a lot of repetitive steps. Examples of suchmethods include isolation of RNA, chromosomal DNA or DNA from plasmidsor other vectors, restriction enzyme digestion of DNA, sequencing ofDNA, elongation and polymerization of DNA by enzymes, oligonucleotidesynthesis, construction of various vectors and gene libraries inmicroorganisms, screening of bacterial strains for biologically activeclones, etc, etc. Each method of this type involves frequent manualhandling of samples like for instance by the transfer of liquids betweencontainers by pipetting, mixing, centrifugation, incubation, washing andprecipitation, just to mention a few procedures.

Another general characteristic of methods in molecular biology is thatsome of the reagents are available only in small amounts or are veryexpensive, and therefore in most cases only very small volumes, usuallyin the μl range, of reagents and reaction mixtures are handled in eachstep of such a method. The reagents or other compounds in the reactionmixtures are moreover often sensitive and therefore special precautionsconsidering temperatures, mechanical sheering, etc, are required. Someof the methods further comprise the handling of potentially hazardouscompounds.

Experimental work in molecular biology is accordingly very timeconsuming and labor intensive. The need for automation can be furtherillustrated with an example of single-stranded template preparation andDNA sequencing, according to a method described in Current Protocols inMolecular Biology, Ed. F. M. Ausbel.

So far automation in molecular biology has mainly been purification andsequencing chemistry have been automated with laboratory roboticequipments essentially automating the same procedures as they areperformed manually Examples of such equipments are: Beckman Biomek 1000and the Zymark robotic system. Although these robots have certainadvantages over the manual methods there are still many drawbacksFirstly, a lot of consumables are used like for instance a new pipettetip for every liquid manipulation. Secondly, the systems are generallaboratory robots and fairly complicated to use and to reprogram.Thirdly, a lot of manual work has to be carried out before as well asafter the robot has been processing a part of the complete method.Sometimes a program has to be interrupted in the middle of a run andsample manually taken out for a centrifugation or incubation step. Thesedrawbacks together with the high cost of a laboratory robot makes it farfrom ideal in a molecular biology laboratory.

Microtiter plates, which are trays having a number of separate reactionwells with openings on one side (the upper side) of the tray, are oftenused for carrying out the manual type of procedures discussed above,since a number of experiments can be carried out in parallel. Samples,liquids etc are introduced into said wells and subjected to the variousprocedures of the particular method. In one type of microtiter platesthere is also an outlet from each well at the bottom side of the platethrough which liquid can be drained from the well to a waste, forinstance by suction. This outlet can be open, but preferably it containsa porous membrane for retaining particles or precipitates in the well,depending on the type of method to be carried out. A microtiter well canalso be packed with a matrix for chromatographic separation of variousconstituents in a sample. Such matrices are mainly of the type in whichcomponents are retained by binding due to electrostatic interaction (ionexchange), hydrophobic interaction or by biospecific affinity, forinstance antigen-antibody interaction, enzyme-substrate interaction,etc. Wells of this type are in fact small chromatography columns.Various types of microtiter plates are manufactured for instance by thecompanies Pall, Costar, Nunc and Millipore.

In U.S. Pat. No. 30,562 discloses an immunological testing device whichcontains a collector structure, a through-passage structure and awashing structure, for safer handling of multiple samples.

U.S. Pat. No. 4,895,706 describes a filter strip assembly comprising afirst strip with a linear array of wells having open top and bottom endsand discrete filter membranes closing the bottom ends, and a secondstrip with wells having closed bottoms placed below the first filterstrip. By the application of a pressure differential across the filtermembrane, such as by vacuum, the wells of the second strip will receivethe filtrate from the respective wells of the first filter strip.

CH-A-669 851 discloses a photometric analyzing apparatus comprising amovable support for a microtiter plate by which the wells of themicrotiter plate may be brought into the beam path of a photometer aswell as below a head member for the introduction into and removal ofwash liquid from the wells. Samples are applied through a movingdispenser arm. The apparatus also comprises an incubator wheremicrotiter plates may be stored vertically, and optionally also astorage station for prepared microtiter plates.

U.S. Pat. No. 4,493,815 discloses a biochemical test plate assembly foruse in both filter assays and fluid retention applications. It comprisesan upper plate with a plurality of discrete apertures, a lower platewith aligned apertures, a microporous film separating the two plates,and a base plate with a central recess to define an enclosed chamberbelow the lower plate, which recess has a vacuum connection.

There is however no system described, as far as we know, which offers aflexible handling of samples with the use of microtiter plates, thelatter forming an excellent basis for a system for performingbiochemical reactions, mainly due to their format and the comparativelyhigh number of wells contained in each plate, and making it possible torun several experiments in parallel and the possibility to include alsovarious matrices for the desired reactions to take place.

It is therefore an object of the present invention to provide anapparatus which offers a simplified handling and processing ofmicrotiter plates or similar processing plates in connection with theperformance of biochemical reactions, a basic concept of the inventionresiding in the provision of special means for the transfer of solutionsor suspensions from the microtiter plate wells, and particularly fromthe wells of one microtiter plate to the wells of another.

According to a particular aspect of the present invention there isprovided an apparatus for automatic handling of microtiter plates,complex liquid handling for providing solutions or suspensions to all orselected wells of a microtiter plate, and means for transferring reagentand sample solutions between microtiter plates, optionally alsoproviding incubation and storage conditions at selected temperatures.

The present invention accordingly relates to an apparatus for carryingout biochemical reactions in microtiter plates comprising

(i) means for receiving and holding a microtiter plate to be processed,said microtiter plate being provided with a plurality of wells and eachwell having a bottom outlet (either a membrane bottom or a capillaryoutlet), and

(ii) a movably mounted device for the application of gas pressure to thewells of said microtiter plate, said device comprising sealing means andbeing movable between a position where it leaves the upper part of themicrotiter plate accessible, and a position where it sealingly contactsthe microtiter plate through said sealing means so that a gas pressuremay be applied to the inlets of said microtiter plate wells for pressingout liquid present therein through the bottom outlet of each well.

In a preferred embodiment, the microtiter plate holding means is capableof holding at least two microtiter plates above each other, the wells ofthe two plates being aligned so that the outlet of a well in the upperplate is placed over the inlet of a corresponding well in the underlyingmicrotiter plate to permit the transfer of liquid from the upper plateto the underlying plate when said gas pressure is applied.

An advantageous development of the apparatus comprises

(i) a processing section comprising the microtiter plate holding means,the gas pressure application device, and optionally means for dispensingliquid to the wells of said upper microtiter plate,

(ii) a storage rack for holding at least two microtiter plates, and

(iii) means for moving microtiter plates between the rack and theprocessing section and optionally the incubation section.

The invention will now be described in more detail, by way of exampleonly, with regard to illustrative embodiments of the invention.Reference will be made to FIGS. 1-4, in which:

FIG. 1 is a schematic view of an embodiment of the apparatus accordingthe invention;

FIG. 2 is a partial sectional view illustrating the transfer means gripmechanism in the apparatus shown in FIG. 1 used for transferringmicrotiter plates from the rack to various sections in the apparatus;

FIG. 3 is a partial sectional view illustrating the gas pressureapplication device of the apparatus in FIG. 1 in contact with amicrotiter plate; and

FIG. 4 is a schematic sectional view of another embodiment of theapparatus according to the invention.

The embodiment illustrated in FIG. 1 comprises the following main parts:a processing section 1; a microtiter plate storage rack 2; transfermeans 3 for moving microtiter plates between the rack 2 and theprocessing section 1; means 4 for dispensing a solution or suspension tothe wells of a first microtiter plate in the processing section 1; apressure device 5 including means for sealingly contacting the devicewith a microtiter plate; an incubation section 6; and optionally means 7for shaking the processing section 1. Control means of the apparatuspreferably include a microprocessor (not shown).

The microtiter plate storage rack 2, in which the various plates neededfor a particular application are loaded, is inserted into a compartment8 in the apparatus. FIG. 2 shows grip means 21, supported on thetransfer means 3 and used for holding a microtiter plate 35 during thetransport between the rack 2 and the processing section 1. The transfermeans 3 can accordingly be moved in the X-direction as well as in the inZ-direction for gripping a microtiter plate in the storage rack 2,moving it out of the rack and down to the level at which it is to bemoved into the processing section 1, or vice versa. (X and Y hereindefine horizontal directions whereas Z is the vertical) The X-directionmovement is indicated in FIG. 2 where a plate 35 is fetched in the rack2 at position X₁ and moved out to the position indicated by X₂ at whichthe Z-direction movement takes place. A microtiter plate can optionallyalso be placed in the incubation section 6 for a predetermined timebefore it is further processed.

In an alternative embodiment of the invention, the storage rack 2 ismoved in a shaft in the instrument so that any microtiter plate can bepositioned at the level where it is to be delivered to the incubationsection 6 or the processing section 1. Transfer means for moving themicrotiter plates to the desired position in this case are analogous tothose described above and will readily be apparent to the man of skillin the art.

The dispensing device 4, which may have several dosing tips, is placedin the processing area 1 and is constructed for adding liquid to thewells. The dispensing device 4 is movable in the X- and Y-directions bybelt drive means 9 and 10, and optionally also in the Z-direction, andcan add liquid to one or more of the wells at a time in anyconfiguration. This dispensing device can be of the type known fromfraction collectors or, in cases when the apparatus is constructed toallow transfer of liquid also between wells in the first microtiterplate, of the type known from autoinjectors. The dispensing device canoptionally also be equipped with means for removing the last drophanging under the dispensing tip 15, for instance by a gas stream.Pumps, for instance peristaltic pumps, supply the dispensing device 4with the desired liquid from buffer or reagent bottles or vials viatubings 11 equipped with valves (not shown).

In order to transfer liquids from a first or upper microtiter plate 35to a second or lower microtiter plate (not shown), the processingsection 1 is provided with the above mentioned gas pressure device 5,preferably comprising a substantially planar plate 12 having at leastthe same length and width as the first microtiter plate 35. In FIG. 1the gas pressure device 5, for clarity reasons, is shown under movementin the X-direction towards its operating position above the firstmicrotiter plate 35.

With reference to FIG. 3, the plate 12 has a collar 31 of an elasticmaterial positioned between bars 37, 38 on the side of plate 12 facingthe first microtiter plate 35. The collar 31 could for instance be aninflatable tubing, for example of silicon rubber, which is connected toa gas pressure source (not shown) via a channel 32. The liquid receivingsecond microtiter plate (not shown) is positioned in respective holdingmeans therefor (also not shown) under the first microtiter plate withthe wells of the two plates aligned so that the outlet of a well in thefirst plate 35 is placed over the inlet of a corresponding well in thesecond microtiter plate.

When the gas pressure device 5 is in its operating position an air tightconnection to the first microtiter plate is obtained so that a space 34is defined by the first microtiter plate 35, the collar 31 and thepressure device plate 12. This is achieved when the two microtiterplates are close to each other and the collar is inflated so that itcontacts the microtiter plate 35. Alternatively, the two plates aresimply pressed together so that the gas tight connection is obtained.

When gas pressure is applied into the space 34 via a channel 33, thesolution in each of the microtiter wells is expelled out of the bottommembrane 36. If the solution from each well is to be directed to wastethis could be achieved by expelling the solutions to a separate wasteplate or to a waste collector 13 at the bottom of the processingsection. Preferably, the microtiter plates in the processing section 1are shaken by the shaking means 7 during processing so that a suspensioncan be kept suspended when the liquid is expelled from the microtiterplate.

For checking the solution expelled from a microtiter plate in theprocessing section, a detector unit 14 is optionally positioned afterthe waste collecting means 13. This is of importance for instance forchecking that enough washing solution has been added to all wells in themicrotiter plate or that a given reaction has taken place.

In order to mix the solutions dispensed to each well of the microtiterplates placed in the processing section 1, at least the first microtiterplate 35 is arranged to be shaken, optionally also when gas pressure isapplied to the wells. This could be achieved by the above mentionedshaking means 7 causing an oscillating movement of the processingsection 1. This feature is also of importance for keeping suspensionssuspended in order to avoid clogging of the filter membrane at the wellbottom.

The flexibility of the above described system is readily appreciated butwill be further illustrated with a general description of its use inbiomolecular applications, however, without limiting the scope of theinvention in any way whatsoever.

The microtiter plates needed for a particular application, that is forexample plates containing the samples to be processed, for instanceafter culture, empty receiving plates or plates containing wellsprepacked with chromatography matrices or a reagent mixture, are placedin the rack 2, which is then positioned in the compartment 8. Buffersolution or reagent bottles are via tubings, pumps and valves connectedto the dosing or dispensing device 4 in the processing area orsection 1. A selected reagent solution can then be pumped into any oneof the wells of a top microtiter plate in the processing section.Reagents can also be supplied from wells of a microtiter plate which isloaded into the storage rack. Such a reagent microtiter plate is fordelivery of solution positioned as the upper or first plate in a stackconfiguration in the processing section as earlier described, and whengas pressure is applied the reagent is expelled to the adjacentmicrotiter plate next below.

Next, the microprocessor is programmed for the actual method to becarried out or an already existing method is called upon or loaded intothe processor. Basic parameters for input into the program are: (i) theposition of each specific microtiter plate in the storage rack, (ii) theselection of plates which should form the stacked combination of platesin the processing section during each step of the procedure to becarried out, (iii) the volume and time for addition of reagents or otherliquids like buffer solutions to a given combination of wells in thefirst microtiter plate, (iv) time and optionally temperatures forincubation and processing, (v) time for transferring solution from wellsin an upper plate to a lower plate or to waste by gas pressure, (vi)shaking time and frequency, as required by the actual application.

FIG. 4 schematically illustrates a simplified embodiment of theinvention. It comprises a box-like frame 41 enclosed by a casing (notshown) and having upper and lower holding means 42 and 43, respectively,for supporting two microtiter plates in a stacked relationship, only amicrotiter plate 44 received by the upper holding means 42 beingillustrated. Of course, further holding means for receiving, say, one ortwo additional microtiter plates may be provided. A compartment 45 isprovided at the bottom of the frame 41 for holding a removable wastecontainer 46. Movably mounted, for example hinged as shown in FIG. 4, tothe top part of frame 41 is a pressure device 47, substantiallyidentical to pressure device 5 shown in FIG. 3. This pressure device 47thus also is of plate configuration and has an inflatable sealing collar48, gas supply channels 49a and 49b, connected to a main supply conduit49, and a gas application space 50. The inflated state of collar 48 isshown in dashed lines. When the pressure device 47 is swung to its"open" position, illustrated by dashed lines in the figure, the wells ofmicrotiter plate 44 are accessible, for example for the addition ofreagent solutions thereto. By swinging the pressure device 47 down tothe "closing" position so that the sealing collar 48 contacts themicrotiter plate 44 and then applying gas pressure, liquid in the wellswill be expelled to the waste container 46 or to the wells of anunderlying microtiter plate in analogous manner as described for theembodiment of FIGS. 1 to 3. Reagent solution may, of course, also inthis case be added to the microtiter plate wells by placing a reagentplate on the upper holding means 42 and the receiver microtiter plate onthe lower holding means 43, and expelling the reagent solution into thewells of the microtiter plate by applying gas pressure to the reagentplate wells by means of the pressure device 47. Optionally, theapparatus in FIG. 4 comprises heating means so that the microtiterplates may be incubated at a desired temperature.

The invention is, of course, not restricted to the embodimentsspecifically described above and shown in the drawings, but manymodifications and changes may be made without departing from the scopeof the inventive concept as defined in the following claims.

What is claimed is:
 1. An apparatus for carrying out biochemicalreactions in microtiter plates which comprises(a) a holder (42, 43) forreceiving and holding a microtiter plate (35; 44) that is provided witha plurality of separate wells, each well having an inlet opening and abottom outlet to expel liquid in the wells, and (b) a movably mounteddevice (5; 47) for applying gas pressure to the wells of said microtiterplate (35; 44), said gas pressure application device (5; 47) comprisinga plate member (12) of at least the same length and width as saidmicrotiter plate (35; 44), said plate member (12) having an inflatablecollar (31; 48) of an elastic material around the periphery facing theperiphery of said microtiter plate and being adapted to sealinglyconnect the gas pressure application device (5; 47) over the microtiterplate so as to thereby form a single gas application space (34; 50)defined by the microtiter plate (35; 44), the inflatable collar (31; 48)and the plate member (12; 47), whereby the same pressure will be quicklyand simultaneously applied to the inlet openings of all wells.